Project 2 takes a clinical and translational genetics approach to identify and characterize genes and their pathways that play a role In the initiation of differentiated thyroid cancer (DTC) for the purposes of the eariiest diagnosis via gene-enabled cancer risk assessment. We will utilize human Cowden syndrome (CS), and a mouse model of human Carney Complex (CNC), as our models epitomizing germline (inherited) predisposition as the first event in initiation in a heritable thyroid neoplasia disorder. CS is a difficult-to- recognize, under-diagnosed heritable disorder characterized by follicular thyroid adenomas (FA), DTC and breast cancer. We found that germline PTEN mutations cause finite subsets of CS and other clinical syndromes, which we collectively term PTEN hamartoma-tumor syndrome (PHTS). Germline PRKR1A mutations associate with CNC. In the first grant period, we have prospectively accrued >3,000 probands from community and academic medical centers who meet CS or CS-like (CSL) criteria and created a web- based PTEN risk calculator based on presence/absence of pathogenic PTEN mutations and clinical characteristics; and showed 32% lifetime risk of DTC in PHTS. We found functional germline variants in SDHB and SDHD, encoding 2 subunits of succinate dehydrogenase, resulting in destabilization of p53 via NQ01 and decreasing ATP levels associated with PTEN nuclear trapping, we developed mouse models of the spectrum of FTC, including FA (thyroid-specific Pten knock-out), locally invasive FTC (Prkaria KO), and metastatic FTC (Pten/Prkaria double KO); preliminary data Indicating downregulation of Sdhb and other Sdh subunits in the FTC models, we broadly hypothesize that Interactions of PTEN, SDHx and PRKR1A play a role in thyroid neoplasia initiation by modulating ROS and other mitochondria-associated energetics. We will (1) analyze SDHx and PRKARIA germline variants In modifying the risk and sub-histology of DTC and of other component cancers in PTEN mutation positive CS/CSL patients; (2) mitochondrial energetics-relevant in vitro functional assays to analyse the interaction of PTEN and SDHx; and (3) physiological validate our human in vivo and in vitro observations in murine models. RELEVANCE (See instructions): Compared to 1 % lifetime risk of developing thyroid cancer, individuals who are at genetic risk carry markedly increased (but never 100%) lifetime risks, often eariy-onset, multifocal and aggressive, and more importantly, almost always carry risks of other extra-thyroldal cancers. However, what remains elusive is the ability to predict which individual carrying predisposition alleles will develop a component malignancy, in this case, DTC. We propose to study energetics for modifying heritable thyroid cancer risk and histology